JP2002136294A - Human kemokine polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide human kemokine polypeptide, to provide a DNA (RNA) encoding the human kemokine polypeptide, and to provide a method for producing the polypeptide by a recombination technology. SOLUTION: A method for using the kemokine polypeptide for the treatments of leukemia, tumor, chronic infection, autoimmune diseases, fibrosis disorders, wound cure and psoriasis is also disclosed. Antagonists against the kemokine polypeptide, and their uses as therapies for treating chronic rheumatoid arthritis, autoimmune diseases, chronic inflammatory diseases, fever not related to infectious diseases, allergic reactions and prostaglandins, and bone marrow failure are also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and also to such polynucleotides and polypeptides. Related to the manufacture of. Above all,
The polypeptides of the present invention are sometimes referred to below as "Ckβ-
4 "and" Ckβ-10 "and are referred to as" chemokines (ch
human chemokine beta-4 and human chemokine beta-10, collectively referred to as "emokine) polypeptides."
The invention also relates to inhibiting the action of such polypeptides.

[0002] Chemokines are an emerging superfamily of small secreted cytokines that are structurally and functionally related. All chemokines show 25-75% homology at the amino acid level and contain spatially conserved cysteine residues, as do the polypeptides of the invention. Peptides that activate neutrophils (N
Members of the "C-X-C branch" (also depending on the position of the first two cysteines in the conserved motif), also known as the (AP) / IL-8 family, are mainly neutrophils. Spheres (eg, IL-8 and NAP
-2), the effect on early inflammatory (pro-inflamma
Although exerting tory activity, members of the "CC branch" family appear to attract certain mononuclear cells. Members of the "CC branch" include PF
4, MIPs, MCPs, and chemokine polypeptides of the present invention.

[0003] Many biological activities have been attributed to this chemokine family. Macrophage inflammatory proteins 1α and 1β are chemotactic for different lymphocyte populations and monocytes (Schch)
all, T.J., Cytokine, 3: 165 (1991))
MCP-1 has been described as a specific monocyte chemoattractant (Matsushima, K. et al., J. Exp. Med., 16
9: 1485 (1989)). A common function of this chemokine family is the ability to stimulate chemotactic migration of different sets of cells, eg, immune cells (leukocytes) and fibroblasts. These chemokines can also activate certain cells in this family.

Because immune cells that respond to chemokines have a vast number of in vivo functions, such chemokine regulation is an important area in the treatment of disease.

[0005] For example, eosinophils destroy parasites and reduce parasite infection. Eosinophils are also responsible for chronic inflammation in the respiratory tract airways. Macrophages serve to suppress tumor formation in vertebrates. In addition, basophils release histamine, which may play an important role in allergic inflammation. Therefore, promoting or inhibiting such cells has broad therapeutic utility.

According to one aspect of the present invention, there are provided novel polypeptides that are Ckβ-4 and Ckβ-10, as well as fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

According to another aspect of the present invention, there is provided a polynucleotide (DNA or RNA) encoding such a polypeptide.

In accordance with yet a further aspect of the present invention, there is provided a method for producing such a polypeptide by recombinant techniques.

In accordance with yet a further aspect of the present invention, such polypeptides, or polynucleotides encoding such polypeptides, are used for therapeutic purposes, eg, in solid tumors, chronic infections, autoimmune diseases, psoriasis. , For treating asthma, allergy, regulating hematopoiesis, and for promoting wound healing.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

According to yet another aspect of the invention, for example, autoimmune diseases, chronic inflammation and infection, histamine-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, Provided are antagonists / inhibitors to such polypeptides that can be used to inhibit the effects of such polypeptides in the treatment of eosinophilia syndrome and pulmonary inflammation.

[0012] These and other aspects of the invention include:
It will be apparent to those skilled in the art from the teachings herein.

The following drawings illustrate embodiments of the present invention and are not intended to limit the scope of the invention, which is encompassed by the claims. FIG. 1 shows that Ckβ-
4 shows the cDNA sequence and the corresponding deduced amino acid sequence. The predicted mature polypeptide comprises 72 amino acids, as the first 24 amino acids represent the predicted leader sequence of Ckβ-4. Use the standard one-letter abbreviations for amino acids. FIG. 2 shows the Ccβ-10 c
1 shows the DNA sequence and the corresponding deduced amino acid sequence. The predicted mature polypeptide comprises 75 amino acids, as the first 23 amino acids represent the predicted leader sequence of Ckβ-10. Use the standard one-letter abbreviations for amino acids. FIG. 3 shows the amino acid sequence homology between Ckβ-4 and the mature peptide of eotaxin (lower). FIG. 4 shows Ckβ-10
1 shows the amino acid sequence homology between (upper) and human MCP-3 (lower).

According to one aspect of the invention, the mature Ckβ-4 polypeptide having the deduced amino acid sequence of FIG. 1, or ATCC Deposit No. 75848 on July 29, 1994.
And the mature Ckβ-10 polypeptide having the deduced amino acid sequence of FIG. 2, or 19
Provided is an isolated nucleic acid (polynucleotide) encoding a mature polypeptide encoded by the cDNA of the clone deposited on July 29, 1994 as ATCC Deposit No. 75849.

[0015] The polynucleotide encoding Ckβ-4 has been found in a cDNA library obtained from human gallbladder. Ckβ-4 is structurally related to the chemokine family. It contains an open reading frame encoding a protein of 116 amino acid residues, of which the first approximately 24 amino acid residues are the predicted leader sequence, so the mature protein has 92 amino acids. Comprising. The protein is
20% for eotaxin over the entire coding sequence
Show the highest degree of homology, with the same identity and 37% similarity. It is also important that four spatially conserved cysteine residues in chemokines are found in the polypeptides of the invention.

A polynucleotide encoding Ckβ-10 was discovered in a cDNA library obtained from 9 weeks of early human tissue. Ckβ-10 is structurally related to the chemokine family. It contains an open reading frame that encodes a protein of 98 amino acid residues, of which the first approximately 23 amino acid residues are a putative leader sequence, so the mature protein has 75 amino acids. Comprising. The protein shows the highest degree of homology to MCP-3 with 65% identity and 77% similarity over the entire coding sequence.

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, wherein the DNA comprises cD
NA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (anti-sense) strand. The coding sequence encoding the mature polypeptide may be the same as the coding sequence shown in FIGS. 1 and 2 or the coding sequence of the deposited clone, or as a result of duplication or degeneracy of the genetic code, It may be a different coding sequence that encodes the same mature polypeptide as the DNA of FIG. 2 and the deposited cDNA.

The polynucleotide encoding the mature polypeptide of FIGS. 1 and 2 or the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence of the mature polypeptide; The coding sequence of the mature polypeptide, and additional coding sequences such as a leader or secretory sequence or a proprotein sequence; the coding sequence of the mature polypeptide (and, optionally, additional coding sequences); and introns of the coding sequence of the mature polypeptide. Or a non-coding sequence such as the non-coding sequence 5 'and / or 3'.

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. I do.

The present invention further relates to the above polynucleotides, which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIGS. 1 and 2 or the cDNA encoded by the cDNA of the deposited clone. Related to the mutant. A variant of the polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Accordingly, the present invention includes a polynucleotide encoding the same mature polypeptide as shown in FIGS. 1 and 2 or the same mature polypeptide encoded by the cDNA of the deposited clone; Variants of such polynucleotides are included which encode fragments, derivatives or analogs of the polypeptides encoded by the polypeptides of FIGS. 1 and 2 or the cDNA of the deposited clone. . Such nucleotide variants include deletion variants,
Substitution variants as well as addition and insertion variants are included.

As indicated above, the polynucleotide may be a naturally occurring allelic variant of the coding sequence shown in FIGS. 1 and 2 or a naturally occurring allelic variant of the coding sequence of the deposited clone. . As is known in the art, an allelic variant is another form of a polynucleotide sequence that can have one or more nucleotide substitutions, deletions or additions, which comprises the encoded polynucleotide. It does not substantially alter the function of the peptide.

The present invention also provides that the coding sequence for the mature polypeptide is a polynucleotide sequence that aids in the expression and secretion of the polypeptide from the host cell, eg, a secretory sequence for controlling transport of the polypeptide from the cell. A functional leader sequence also includes a polynucleotide that can be fused in the same reading frame. A polypeptide having a leader sequence is a preprotein and may have a leader sequence that has been cleaved by a host cell to form a mature polypeptide. The polynucleotide may also encode a proprotein that is a mature protein with additional 5 'amino acid residues. A mature protein having a prosequence is a proprotein and an inactive form of the protein. Once the prosequence is cleaved, the active mature protein remains.

Thus, for example, the polynucleotide of the present invention may encode a mature protein, or a protein having a prosequence, or a protein having both a prosequence and a presequence (leader sequence).

The polynucleotide of the present invention may also have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the present invention. In the case of a bacterial host, the marker sequence may be a hexa-histidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker, or, for example, A mammalian host, e.g., CO
When using S-7 cells, the marker sequence is:
It may be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell, 37:76).
7 (1984)).

The present invention further provides that at least 50
%, And preferably 70% identity, to polynucleotides that hybridize to the above sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the term "stringent conditions" refers to at least 95%
It also preferably means that hybridization will only occur if there is at least 97% identity. In a preferred embodiment, the polynucleotide that hybridizes to the above-described polynucleotide has substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of FIGS. 1 and 2 or the deposited cDNA. Encodes a retained polypeptide.

The deposit referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. These deposits are provided merely as a convenience to those skilled in the art, and
It is not an admission that it is required under U.S.C. §112. The sequence of the polynucleotide contained in the deposited material, and also the amino acid sequence of the polypeptide encoded thereby, form part of the present specification and may include any of the sequences herein. Whenever it conflicts with the description, it is checked. A license may be required to make, use, or sell the deposited material, and no such license is granted herein.

The present invention further provides a chemokine polypeptide having the deduced amino acid sequence of FIGS. 1 and 2 or having the amino acid sequence encoded by the deposited cDNA, and also fragments of such a polypeptide. , Analogs and derivatives.

When referring to the polypeptides of FIGS. 1 and 2 or the polypeptide encoded by the deposited cDNA, the terms "fragment,""derivative," and "analog" refer to such polypeptides substantially. Polypeptides that have the same biological function or activity. Thus, analogs include protein that can be activated by cleavage of the protein portion to produce an active mature polypeptide.

[0030] The chemokine polypeptide of the present invention may be a recombinant, natural or synthetic polypeptide, preferably a recombinant polypeptide.

Fragments, derivatives or analogs of the polypeptides of FIGS. 1 and 2 or the deposited cDNA may include (i) one or more amino acid residues having the same or different Residues (preferably, isomorphic amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code. (Ii) one or more amino acid residues containing a substituent, or (ii)
i) the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (eg, polyethylene glycol); or (iv) a leader or secretory sequence, or the mature polypeptide Additional amino acids may be fused to the mature polypeptide, such as a sequence used for purification of a protein, or a protein sequence. Such fragments, derivatives and analogs are deemed to be within the skill of the art in light of the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the substance has been removed from its original environment (eg, the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide in a living animal has not been isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system has been isolated. ing. Such polynucleotides can be part of a vector, and / or such polynucleotides or polypeptides can be part of a composition, and such vectors or compositions are not part of their natural environment. And still isolated.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and recombinant production of the polypeptides of the present invention.

The host cell is genetically engineered (transduced or transformed or transfected) with a vector of the invention, which may be, for example, a cloning vector or an expression vector.
The vector can be, for example, in the form of a plasmid, virus particle, phage, and the like. The engineered host cell activates the promoter, selects for the transformant,
Alternatively, it can be cultured in a conventional nutrient medium that has been modified to be suitable for amplifying the Ckβ-4 and Ckβ-10 genes. Culture conditions such as temperature, pH, etc. are those previously used in the host cell selected for expression and will be apparent to those skilled in the art. The polynucleotides of the present invention can be used to produce peptides by recombinant techniques. Thus, for example, the polynucleotide can be included in any one of a variety of expression vectors for expressing the polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, eg, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors obtained from a combination of plasmid and phage DNA; vaccinia, adenovirus , Fowlpox virus, pseudorabies, and the like. However, any other vector can be used as long as it is replicable and viable in the host.

[0036] The appropriate DNA sequence can be inserted into the vector by various methods. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. Such and other methods are:
It is believed to be within the purview of those skilled in the art.

The DNA sequence of the expression vector is operably linked to an appropriate expression control sequence (promoter), and
Let the composition take place. As representative examples of such promoters include the following:. LTR or SV40 promoter, E. coli lac or trp, the phage lambda P _L promoter, and expression of genes in prokaryotic or eukaryotic cells or their viruses Other promoters known to control. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, expression vectors may be such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli .
It is preferred to include one or more selectable marker genes to confer phenotypic characteristics for the selection of transformed host cells.

A suitable DNA sequence, as described above, as well as a vector containing a suitable promoter or control sequence, can be used to transform a suitable host to enable that host to express the protein. Can be

Representative examples of suitable hosts include the following: E. coli , Streptomyces , Salmonella ty
bacterial cells such as phimurium ; fungal cells such as yeast; insect cells such as Drosophila and Sf9 ; animal cells such as CHO, COS or Bowes melanoma; The selection of a suitable host will be within the skill of the art in light of the teachings herein.

In particular, the present invention also includes a recombinant construct comprising one or more of the sequences broadly described above. The construct comprises a vector, such as a plasmid or viral vector, into which the sequences of the invention have been inserted in forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter operably linked to the sequence. Many suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are given as examples. For bacteria: pQE70, pQE
60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript
SK, pbsks, pNH8A, pNH16a, pNH18A, p
NH46A (Stratagene); ptrc99a, pKK223-
3, pKK233-3, pDR540, pRIT5 (Pharm
acia). For eukaryotes: pWLNEO, pSV2CAT, pO
G44, pXT1, pSG (Stratagene), pSVK3, p
BPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used as long as they are replicable and viable in the host.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or another vector having a selection marker. Two suitable vectors are PKK232-8 and PCM7. Individually named bacterial promoters include lacI, lacZ,
T3, T7, gpt, lambda _P R, include _{P L} and trp. Eukaryotic promoters include CMV immediate early
early), HSV thymidine kinase, early and late S
V40, LTR from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further aspect, the present invention relates to a host cell comprising the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into host cells can be by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation. (Davis, L., Di
bner, M., Battey, L., Basic Methods in Molecula
r Biology (1986)).

The construct in the host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptide of the present invention can be produced synthetically using a conventional peptide synthesizer.

[0044] The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Such a protein,
Cell-free translation systems can also be used for production with RNA obtained from the DNA constructs of the present invention. Cloning and expression vectors suitable for use in prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular.
Cloning: A Laboratory Manual, Second Edition, Cold S
Spring Harbor, New York, (1989), the disclosure of which is hereby incorporated by reference.

DNA encoding the polypeptide of the present invention
Transcription by higher eukaryotes is increased by inserting an enhancer sequence into the vector. The enhancer is
Acts on a promoter to increase its transcription, usually
It is a cis-acting element of DNA of about 10-300 bp.
Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers at bp 100-270.

Typically, the recombinant expression vector contains an origin of replication and a selectable marker that allows for transformation of the host cell, such as the ampicillin resistance gene of E. coli and the S. cerevisiae TRP1 gene, and the downstream gene. A promoter derived from a highly expressed gene to drive transcription of the structural sequence would be included. Such promoters include, inter alia, 3
-It can be obtained from operons encoding glycolytic enzymes such as phosphoglycerate kinase (PGK), α-factor, acid phosphatase or heat shock proteins. The heterologous structural sequence is assembled in appropriate phase, with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. . Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that confers desired properties, such as stabilization or purification of the expressed recombinant product.

[0047] Expression vectors useful for use in bacteria include structural DNA sequences encoding desired proteins.
It is constructed by insertion into an operable reading phase having a functional promoter, with appropriate translation initiation and termination signals. The vector includes one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification in the host. Will be. Prokaryotic hosts suitable for transformation include E. coli , Bacillus subtilis , Salmonella typ.
himurium , genus Pseudomonas, genus Streptomyces,
And various species within the genus Staphylococcus, but others can also be used as selection agents.

As a representative, but non-limiting example, a vector useful for use in bacteria can be obtained from a commercially available plasmid comprising the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). It may comprise a selectable marker and a bacterial origin of replication. Such commercially available vectors include, for example, pKK2
23-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madi)
son, WI, USA). These pBR322 "bone nuclei" portions are combined with an appropriate promoter and the structural sequence to be expressed.

After transforming a suitable host strain and growing the host strain to a suitable cell density,
The selected promoter is induced by a suitable method (eg, temperature shift or chemical induction) and the cells are cultured for an additional period.

The cells are generally collected by centrifugation and disrupted by physical or chemical methods, and the resulting crude extract is retained for further purification.

The microbial cells used for protein expression can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. It is well known to those skilled in the art.

[0052] A variety of mammalian cell culture systems can also be used to express the recombinant protein.
Examples of mammalian expression systems include Gluzman, Cell, 23: 1.
75 (1981), and the COS-7 line of monkey kidney fibroblasts, and other cell lines capable of expressing compatible vectors, such as C12
7, 3T3, CHO, HeLa and BHK cell lines are included. Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed sequences flanking the 5 '. Will also comprise.
The DNA sequence obtained from SV40 splicing, and the polyadenylation site can be used to provide the required non-transcribed genetic elements.

Chemokine polypeptides can be prepared by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography,
It can be recovered from the recombinant cell culture and purified by methods including affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. If desired, a protein regeneration step can be used to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

The chemokine polypeptides of the present invention can be naturally purified products, or products of chemical synthesis, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, It can be produced by recombinant technology (by insect and mammalian cells). Depending on the host used in the recombinant production method, the polypeptide of the present invention may be glycosylated or not glucosylated. The polypeptides of the present invention may also include a first methionine amino acid residue.

Chemokine polypeptides can be used to inhibit bone marrow stem cell colonization during cancer chemotherapy and as an adjunct protective treatment against leukemia.

The chemokine polypeptides can also be used to inhibit epidermal keratinocyte proliferation as a treatment for psoriasis characterized by keratinocyte hyperproliferation.

The chemokine polypeptides can also be used to treat solid tumors by stimulating the entry and activation of host defense cells, such as cytotoxic T cells and macrophages. They can also be used to increase host defense against resistant chronic infections, for example, mycobacterial infection by attracting and activating mycobacterial leukocytes.

The chemokine polypeptide also comprises IL2
It can also be used to treat autoimmune diseases and lymphocytic leukemia by inhibiting the proliferation of T cells by inhibiting the biosynthesis of.

Ckβ-4 and Ckβ-10 can also be released by recruiting inflammatory cells that clear debris and promote connective tissue, and by controlling fibrosis mediated by excess TGFβ. It can also be used in wound healing. In the same way, Ckβ-
4 and Ckβ-10 can also be used to treat other fibrotic disorders, including cirrhosis, osteoarthritis and pulmonary fibrosis. The chemokine polypeptides also increase the presence of eosinophils, which have a characteristic function of killing parasite larvae that invade tissues, such as schistosomiasis, trichinellosis and ascariasis. They can also be used to regulate hematopoiesis by regulating the activation and differentiation of various hematopoietic progenitor cells.

[0060] Chemokine polypeptides can also be used as inhibitors of angiogenesis and, therefore, they have antitumor effects.

The chemokine polypeptide of the present invention also includes
It is also useful for identifying other molecules with similar biological activity. Examples of screening for this are:
Isolating the coding region of a gene by synthesizing an oligonucleotide probe using a known DNA sequence. Labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention, human cDNA,
Used to screen libraries of genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention also relates to diagnostic assays for quantitatively and qualitatively detecting altered levels of a polypeptide, or mRNA that provides a message for such a polypeptide. Such assays are well known in the art and include ELISA assays, radioimmunoassays and RT-PCR. The polypeptides detected in the assay, or their polypeptides.
mRNA levels can be used to elucidate the significance of the polypeptide in various diseases and to diagnose diseases in which altered levels of the polypeptide may be important.

The present invention provides a method for identifying a receptor for a polypeptide. The gene encoding the receptor is
It can be identified by expression cloning. Polyadenylation RNA is prepared from cells that respond to the polypeptide, and cDNA libraries made from this RNA are divided into pools and used to transfect COS cells or other cells that do not respond to the polypeptide. . Transfected cells grown on glass slides are exposed to the labeled polypeptide. The polypeptide can be labeled by a variety of methods, including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified, subpools are prepared and re-transfected using iterative subpooling and rescreening methods, ultimately resulting in a single clone encoding the putative receptor. Other methods for identifying the receptor can include binding the labeled polypeptide to the cell membrane with photoaffinity or extracting a preparation that expresses the receptor molecule. Bridge
The (cross-linked) material is separated by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptor for the polypeptide can be excised, split into peptide fragments, and subjected to protein microsequencing. Using the amino acid sequence obtained from microsequencing, a set of oligonucleotide probes will be designed and a cDNA library will be screened to identify the gene encoding the putative receptor.

The present invention is directed to enhancing the interaction of polypeptides with their identified receptors (agonists),
Alternatively, methods for screening drugs to identify blocking (antagonist) drugs are provided. An agonist is a compound that increases the natural biological function of the polypeptide, whereas an antagonist eliminates such function. As an example, a mammalian cell or membrane preparation that expresses a receptor for the polypeptide will be incubated with a labeled chemokine polypeptide, eg, radioactivity, in the presence of a drug. The ability of the drug to enhance or block this interaction could then be measured.

[0065] Potential antagonists include antibodies,
In some cases, it also includes oligonucleotides that bind to the polypeptide. Another example of a potential antagonist is a negative dominant mutant of the polypeptide. Negative dominant mutants are polypeptides that bind to the receptor for the wild-type polypeptide but do not retain biological activity.

Assays for detecting negative dominant mutants of a polypeptide use a multiwell chemotaxis chamber with a polycarbonate membrane without polyvinylpyrrolidone to detect potential antagonists / inhibitors or An in vitro chemotaxis assay that measures the chemoattractant ability of a polypeptide on leukocytes in the presence and absence of an agonist molecule is included.

[0067] Antisense constructs made using antisense technology are also potential antagonists. Antisense technology can be used to control gene expression by triple helix formation or antisense DNA or RNA, both of which are based on the binding of polynucleotides to DNA or RNA. For example, encoding a mature polypeptide of the invention,
The 5 'coding portion of the polynucleotide sequence is used to design an antisense RNA oligonucleotide approximately 10 to 40 base pairs in length. DNA oligonucleotides were designed to be complementary to the gene region involved in transcription (triple helix-Lee et al., Nucl. Acids Res., 6: 3).
073 (1979); Cooney et al., Science, 241:
456 (1988); and Dervan et al., Science, 2
51: 1360 (1991)), thereby preventing transcription and production of the polypeptide. Antisense RNA oligonucleotides are
Hybridizes to RNA and blocks translation of mRNA molecules into polypeptides (antisense-Okano,
J. Neurochem., 56: 560 (1991); Oligode.
oxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1
988)). Since the above oligonucleotides are also delivered to cells, antisense RNA or DNA can be expressed in vivo to inhibit polypeptide production.

Another potential antagonist, which has lost its biological function, but nevertheless recognizes and binds to a receptor for a polypeptide, thereby activating the receptor A peptide derivative of a polypeptide that is a naturally or synthetically modified analog of a polypeptide that blocks the polypeptide. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.

The antagonists are useful in autoimmune diseases and chronic inflammatory diseases and infectious diseases, of macrophages and their precursors, and of neutrophils, basophils,
B lymphocytes and some T cell subsets, such as
It can be used to inhibit the chemotaxis and activation of activated and CD8 cytotoxic T cells, as well as natural killer cells. Examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and insulin-dependent diabetes. Some infections include silicosis due to preventing recruitment and activation of mononuclear phagocytes, sarcoidosis, idiopathic pulmonary fibrosis, idiopathic hypereosin-prone syndrome due to preventing eosinophil production and migration, Includes endotoxin shock by preventing macrophage migration and production of the chemokine polypeptides of the invention. The antagonists can also be used to treat atherosclerosis by preventing monocyte infiltration in the arterial wall.

The antagonists can also be used to treat histamine-mediated allergic reactions by inhibiting chemokine-induced mast cell and basophil degranulation and histamine release. .

The antagonists can also be used to treat inflammation by preventing the attraction of monocytes to the wound area. They can also be used to regulate normal lung macrophage numbers, as acute and chronic inflammatory pulmonary diseases are associated with mononuclear phagocyte osteogenesis in the lung.

Antagonists can also be used to treat rheumatoid arthritis by preventing the attraction of monocytes into the synovial fluid in the patient's joints. Monocyte influx and activation plays an important role in the pathogenesis of both degenerative and inflammatory arthritis.

The antagonists also interfere with the biosynthesis of other inflammatory cytokines, mainly IL-1 and T
It can also be used to prevent deleterious cascades caused by NF. In this way, the antagonist can also be used to prevent inflammation. The antagonists can also be used to block prostaglandin-independent fever induced by chemokines.

The antagonists can also be used to treat diseases of bone marrow failure, such as aplastic anemia and myelodysplastic syndrome.

The antagonists can also be used to treat asthma and allergy by preventing eosinophil accumulation in the lung. The antagonist can also be used in a composition with a pharmaceutically acceptable carrier, for example, a carrier as described below.

The chemokine polypeptides and agonists or antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. In connection with such containers, a notice may be provided in the form prescribed by governmental authorities that regulate the manufacture, use or sale of pharmaceutical or biological products, the notice being provided for administration to humans. Represents the approval of the relevant bureau of manufacture, use or sale for Further, the polypeptides of the present invention can be used with other therapeutic compounds.

The pharmaceutical composition may be topical, intravenous, intraperitoneal,
Administration can be by any convenient method, such as intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. The polypeptide is administered in an amount effective to treat and / or prevent the particular indication. Generally, the polypeptide is administered in an amount of at least about 10 μg / kg (body weight),
Most often they are about 8 mg / kg (body weight) per day
Will not be administered. Most often,
The dosage is about 10 μg / kg to about 1 mg / kg (body weight) daily, taking into account the administration route and symptoms.

The chemokine polypeptides and agonists or antagonists can be used in accordance with the present invention due to the in vivo expression of such polypeptides, often referred to as “gene therapy”.

Thus, for example, cells from a patient are engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells are then provided to a patient to be treated with the peptide. Such methods are well-known in the art. For example, cells can be manipulated by methods known in the art by use of retroviral particles comprising RNA encoding a polypeptide of the present invention.

Similarly, cells can be engineered in vivo for expression of the polypeptide in vivo, eg, by methods known in the art. As is known in the art, producing cells for producing retroviral particles comprising RNA encoding a polypeptide of the invention, in order to treat the cells in vivo and express the polypeptide in vivo. It can be administered to a patient. These and other methods for administering a polypeptide of the present invention by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, an expression vehicle for manipulating cells may be other than a retrovirus, for example, an adenovirus that can be used to manipulate cells in vivo after being combined with a suitable delivery vehicle.

The sequences of the present invention are also valuable for chromosome identification. The sequence can be specifically targeted to a particular location on an individual human chromosome and hybridized. Moreover, there is currently a need to identify specific sites on the chromosome. Chromosome marking reagents based on actual sequence data (repetitive polymorphisms) are currently scarcely available for marking chromosomal locations. D according to the invention
Mapping NAs to chromosomes is an important first step in relating their sequences to genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing cDNA-derived PCR primers (preferably 15-25 bp). cDNA
Using computer analysis of genomic DNA
The rapid selection of primers that do not span more than one exon complicates the amplification process.
These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will give the amplified fragment. PCR mapping of somatic cell hybrids
It is a quick method for assigning a specific DNA to a specific chromosome. Utilizing the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar manner using a panel of fragments from a pool of specific chromosomes or large genomic clones. Other mapping methods that can also be used to map to the chromosome include in situ hybridization, labeled flow-sorted (f
low-sorted) chromosomes and preselection by hybridization to construct a chromosome-specific cDNA library.

Fluorescence in situ hybridization (FIS) of cDNA clones to metaphase chromosomal spreads
Using H), the exact chromosome location can be provided in one step. This technique can be used with cDNAs as short as 500 or 600 bases;
Clones larger than 00 bp are more likely to bind to unique chromosomal locations that have sufficient signal intensity for easy detection. FISH requires the use of clones from which ESTs are obtained, and the longer the better. For example, 2,0
00bp is better, 4000 is better,
More than 4,000 is probably not necessary to get good results at a reasonable time rate. A review of this technique requires Verma
Et al., Human Chromosomes: a Manual of Basic Tech
niques, Pergamon Press, New York (1988)
See

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be associated with genetic map data. Such data is
For example, V. McKusick, Mendelian Inheritance in
Man (Johns Hopkins University Welch Medical)
(Available online via Library). The relationship between the gene mapped to the same chromosomal region and the disease is then confirmed by binding analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is found in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be disease-causing.

At present, from physical and genetic mapping analyses, a cDNA that is correctly localized to a chromosomal region associated with a disease could be one of 50-500 possible causative genes. . (This assumes a 1 megabase mapping analysis and one gene per 20 kb).

[0088] Antibodies to the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as immunogens to produce them. These antibodies are
For example, it can be a polyclonal or monoclonal antibody. The invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the products of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments.

Antibodies raised against polypeptides corresponding to the sequences of the present invention can be obtained by injecting the polypeptides directly into animals or by administering the polypeptides to animals, preferably non-human animals. Can be. The antibody so obtained will then bind to the polypeptide itself. In this way, even sequences that encode only fragments of the polypeptide,
It can be used to produce antibodies that bind the whole naturally occurring polypeptide. Such an antibody can then be used to isolate the polypeptide from tissues expressing the polypeptide.

For preparing monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology
(Kohler and Milstein, 1975, Nature, 25
6: 495-497), trioma technology, human B
Cell hybridoma technology (Kozbor et al., 1983, Immun
ology Today 4:72), and EBV-hybridoma technology (Cole) for producing human monoclonal antibodies.
Et al., 1985, Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).

The techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to the immunogenic polypeptide products of the invention.

The present invention is further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts are by weight unless otherwise indicated.

To facilitate understanding of the following examples,
Some frequently occurring methods and / or terms are described.

"Plasmid" is a lowercase p
And / or followed by capital letters and / or numbers. The starting plasmids herein are commercially available, publicly available on a non-limiting basis, or can be constructed from plasmids available by published methods. In addition, plasmids equivalent to the described plasmids are known in the art and will be apparent to those skilled in the art.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to one of skill in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, DN
Digest 5-50 μg of A with 20-250 units of enzyme.
Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 ° C. are usually utilized, but can be varied according to the supplier's instructions. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

The size separation of the cleaved fragments
oeddel, D et al., Nucleic Acids Res., 8: 4057.
Performed using 8% polyacrylamide gel as described by (1980).

“Oligonucleotide” is a single-stranded polydeoxynucleotide that can be chemically synthesized.
Or two complementary polynucleotide strands. Since such a synthetic oligonucleotide does not have a 5 'phosphate, it will not ligate to another oligonucleotide without adding a phosphate with ATP in the presence of a kinase. Synthetic oligonucleotides are
Will ligate to fragments that have not been dephosphorylated.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T. et al., Ibid., P. 146). Unless otherwise stated, ligation was performed with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amount of the DNA fragment to be ligated.
This can be accomplished using known buffers and conditions.

Unless otherwise stated, transformations were performed according to Graham, F. and Van der Eb, A., Vi.
rology, 52: 456-457 (1973).

[0100]

EXAMPLES Example 1 Bacterial Expression and Purification of Ckβ-4 First, a DNA sequence encoding Ckβ-4, ATCC
No. 75848 is amplified utilizing PCR oligonucleotide primers corresponding to the 5 ′ and 3 ′ sequences of the processed Ckβ-4 protein (no putative signal peptide sequence). Additional nucleotides corresponding to Ckβ-4 were added to the 5 ′ and 3 ′ sequences, respectively.
The 5 'oligonucleotide primer has the sequence: And contains the SphI restriction enzyme site (bold print) followed by a 17 nucleotide Ckβ-4 coding sequence (underlined print) beginning at the second nucleotide of the mature protein coding sequence. The ATG codon is included in the SphI site. In the next codon following ATG, the first base is from the SphI site and the remaining two bases correspond to the second and third bases of the first codon of the putative mature protein . As a result, the first base at this codon changes from G to C compared to the original sequence, resulting in the replacement of E by Q in the recombinant protein. 3 'sequence: Contains the sequence complementary to the BamH1 site (boldface type) followed by a 21 nucleotide gene-specific sequence before the stop codon. The restriction enzyme site is located in the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Av).
e., Chatsworth, CA 91311). pQE-70 is resistant to antibiotics (Amp ^r ),
The bacterial origin of replication (ori), the IPTG-regulatable promoter operator (P / O), the ribosome binding site (R
BS), a 6-His tag and a restriction enzyme site. Then, pQE-70 was digested with SphI and BamH1. The amplified sequence was ligated into pQE-70 and inserted in frame with the sequence encoding the histidine tag and RBS. FIG. 8 illustrates this arrangement.
2 shows a schematic display of. The ligation mixture is then used to create a sample, as described in Sambrook, J. et al., Molecular Cloni.
ng: A Laboratory Manual, Cold Spring Laborat
ory Press (1989)
E. coli available under the trademark M15 / rep4 from Qiagen .
The strain was transformed. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ). Transformants were identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Colonies containing the desired construct were grown overnight in liquid culture in LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml).
(O / N). The O / N culture was used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells
It grew to an optical density of 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG ("isopropyl-BD-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTG induces P / O clearing and increases gene expression by inactivating the lacI repressor. Cells were grown for an additional 3-4 hours. The cells were then collected by centrifugation. The cell pellet is treated with the chaotropic agent 6 mol of guanidine HC.
solubilized in l. After clarification, the solubilized Ckβ-4 was purified from this solution by chromatography on a nickel-chelate column under conditions that allowed strong binding by the protein containing the 6-histidine tag. (Hoc
huli, E. et al., J. Chromatography 411: 177-1.
84 (1984)). 6 moles of guanidine HCl (pH 5.
In 0), Ckβ-4 (purity> 98%) was eluted from the column. Regeneration of the protein from GnHCl can be accomplished by several protocols. (Jaenicke, R.
And Rudolph, R., Protein Structure-A Pract
ical Approach, IRL Press, New York (19
90)). First, GnHCL using step dialysis
Is removed. Alternatively, the purified protein isolated from the Ni-chelate column is replaced with reduced linear GnHCL.
It can be attached to a second column through a gradient. After regeneration of the protein while binding to the column, 250 mM imidazole, 150 mM NaCl, 2.
Elute with a buffer containing 5 mM Tris-HCl (pH 7.5) and 10% glycerol. Finally, the soluble protein is dialyzed against a storage buffer containing 5 mM ammonium bicarbonate.

Example 2 Bacterial Expression and Purification of Ckβ-10 First, the DNA sequence encoding Ckβ-10, ATC
C-75849, processed Ckβ-10
5 'and 3' of protein (without signal peptide sequence)
The sequence is amplified using PCR oligonucleotide primers corresponding to the sequence and the vector sequence on the 3 ′ side of the Ckβ-10 gene. Additional nucleotides corresponding to Ckβ-10 were added to the 5 ′ and 3 ′ sequences, respectively. The 5 'oligonucleotide primer has the sequence: And contains the SphI restriction enzyme site (bold print), followed by a 19 nucleotide Ckβ-10 coding sequence (underlined print) beginning with the sequence encoding the mature protein. The ATG codon is included in the SphI site. 3 'sequence: Contains the sequence complementary to the BamH1 site (bold print) followed by a 19 nucleotide gene-specific sequence before the stop codon. The restriction enzyme site is located in the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Av).
e., Chatsworth, CA 91311). pQE-70 is resistant to antibiotics (Amp ^r ),
The bacterial origin of replication (ori), the IPTG-regulatable promoter operator (P / O), the ribosome binding site (R
BS), a 6-His tag and a restriction enzyme site. Then, pQE-70 was digested with SphI and BamH1. The amplified sequence was ligated into pQE-70 and inserted in frame with the sequence encoding the histidine tag and RBS. FIG. 10 shows a schematic representation of this arrangement. The ligation mixture is then used to create a sample, as described in Sambrook, J. et al., Molecular Cloning: A Labo.
ratory Manual, Cold Spring Laboratory Press
(1989) was transformed into a strain of E. coli available under the trademark M15 / rep4 from Qiagen. M15 / rep4 is plasmid pREP4
Which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ). Transformants were identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Colonies containing the desired construct were grown overnight in liquid culture in LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml) (O /
N). The O / N culture was used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells
It grew to an optical density of 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG ("isopropyl-BD-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTG induces P / O clearing and increases gene expression by inactivating the lacI repressor. Cells were grown for an additional 3-4 hours. The cells were then collected by centrifugation. The cell pellet is treated with the chaotropic agent 6 mol of guanidine HC.
solubilized in l. After clarification, the solubilized Ckβ-10 was purified from this solution by chromatography on a nickel-chelate column under conditions that allowed strong binding by the protein containing the 6-histidine tag. (H
ochuli, E. et al., J. Chromatography 411: 177-.
184 (1984)). 6 moles of guanidine HCl (pH
5.0), Ckβ-10 (purity> 98%) was eluted from the column. Regeneration of the protein from GnHCl can be accomplished by several protocols. (Jaenick
e, R. and Rudolph, R., Protein Structure-A
Practical Approach, IRL Press, New York (1990)). First of all, using stage dialysis, Gn
Remove HCL. Alternatively, the purified protein isolated from the Ni-chelate column is converted to reduced linear Gn.
It can be bound to a second column through an HCL gradient. After regeneration of the protein while binding to the column, 250 mM imidazole, 150 mM Na
Cl, 25 mM Tris-HCl (pH 7.5) and 10%
Elute with a buffer containing glycerol. Finally, the soluble protein is dialyzed against a storage buffer containing 5 mM ammonium bicarbonate. The protein was then analyzed on an SDS-PAGE gel.

Example 3 Expression of recombinant Ckβ-4 expression plasmid and Ckβ-4 HA in COS cells is as follows: 1) SV40
Replication origin, 2) ampicillin resistance gene, 3) E.col
i) Origin of replication, 4) Vector pcDNAI / Amp (Invitrogen) containing CMV promoter followed by polylinker region, SV40 intron and polyadenylation site
Obtained from DNA encoding the complete Ckβ-4 precursor and the HA tag fused in frame to its 3 ′ end
Since the fragment was cloned into the polylinker region of the vector, recombinant protein expression was
Directed under the V promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as described above (I. Wilson, H. Niman,
R. Heighten, A. Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). By fusing the HA tag to the target protein, the recombinant protein can be easily detected with an antibody that recognizes the HA epitope. The plasmid construction method is described below: DNA sequence encoding Ckβ-4, ATC
C-th No. was constructed by PCR on the original EST, cloned using two primers: 5 ′ primer Is the HindIII site followed by 20 starting from the start codon.
Including the nucleotide Ckβ-4 coding sequence; 3 ′ sequence Is the sequence complementary to the XbaI site, a translation stop codon,
Includes the HA tag, and the last 20 nucleotides of the Ckβ-4 coding sequence (not including the stop codon). Thus, the PCR product contains a HindIII site, a Ckβ-4 coding sequence, followed by an HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XbaI site. P
The DNA fragment and the vector, pcDNAI / Amp, amplified by CR were digested with HindIII and XbaI restriction enzymes and ligated. The ligation mixture was transferred to E. coli strain SURE (Stratagene Cloning S.
ystems, 11099 North Torrey Pines Road, L
a, available from Jolla, CA 92037) and the transformed cultures were placed on ampicillin media plates to select for resistant colonies. Plasmid DNA was isolated from the transformants and tested by restriction analysis for the presence of the correct fragment. For expression of recombinant Ckβ-4, D
COS cells were transfected with the expression vector by the EAE-DEXTRAN method (J. Sambrook, EF
ritsch, T. Maniatis, Molecular Cloning: A Lab
oratory Manual, Cold Spring Laboratory Pres
s, (1989)). Expression of Ckβ-4 HA protein was detected by radiolabeling and immunoprecipitation. (E.
Harlow, D. Lane, Antibodies: A Laboratory Ma
nual, Cold Spring Laboratory Press, (198
8)). Two days after transfection, cells were ^harvested at ³⁵
Labeled with S-cysteine for 8 hours. Then, the cell culture medium was collected and the cells were washed with detergent (RIPA buffer (150 mM).
M NaCl, 1% NP-40, 0.1% SDS, 1%
NP-40, 0.5% DOC, 50 mM Tris, pH
The cells were lysed in 7.5)). (Wilson, I. et al., Ibid. 37:76.
7 (1984)). Both cell lysate and culture medium were sedimented with monoclonal antibodies specific for HA. The precipitated proteins were analyzed by SDS-PAGE.

Example 4 Expression of recombinant Ckβ-10 expression plasmid, Ckβ-10 HA in COS cells is as follows: 1) SV40
Replication origin, 2) ampicillin resistance gene, 3) E.co.
li Replication origin, 4) Vector pcDNAI / Amp (Invitrogen) containing CMV promoter followed by polylinker region, SV40 intron and polyadenylation site.
Obtained from Complete Ckβ-10 precursor and its 3 ′
DN encoding HA tag (tag) fused in frame at the end
Since the A fragment was cloned into the polylinker region of the vector, recombinant protein expression was
Directed under the MV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as described above (I. Wilson, H. Niman,
R. Heighten, A. Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). By fusing the HA tag to the target protein, the recombinant protein can be easily detected with an antibody that recognizes the HA epitope. The plasmid construction method is described below: DNA sequence encoding Ckβ-10, AT
CC 75849 was constructed by PCR on the original EST, cloned using two primers: 5 ′ primer Is the HindIII site, followed by the start codon 19
Including the nucleotide Ckβ-10 coding sequence; 3 ′ sequence Is the sequence complementary to the XbaI site, a translation stop codon,
HA tag, and the last 19 of the Ckβ-10 coding sequence.
Nucleotides (not including the stop codon). Thus, the PCR product contains a HindIII site, a Ckβ-10 coding sequence, followed by an HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XbaI site. The PCR amplified DNA fragment and vector, pcDNAI / Amp, were converted to HindIII and BamH.
Digested with 1 restriction enzyme and ligated. The ligation mixture was transferred to the E. coli strain SURE (Stratagene Cl
oning Systems, 11099 North Torrey Pines R
oad, available from La Jolla, CA 92037) and the transformed cultures were placed on ampicillin media plates to select for resistant colonies. Plasmid DNA was isolated from the transformants and tested by restriction analysis for the presence of the correct fragment. Recombinant Ckβ-10 was expressed by the DEAE-DEXTRAN method using CO
S cells were transfected with an expression vector (J. Sam
brook, E. Fritsch, T. Maniatis, Molecular Clon
ing: A Laboratory Manual, Cold Spring Labora
tory Press, (1989)). Expression of Ckβ-10 HA protein was detected by radiolabeling and immunoprecipitation. (E. Harlow, D. Lane, Antibodies: A Labo
ratory Manual, Cold Spring Laboratory Press,
(1988)). Two days after transfection, cells were labeled with ³⁵ S-cysteine for 8 hours. Then
Collect cell culture medium and remove cells with detergent (RIPA buffer
(150 mM NaCl, 1% NP-40, 0.1% SD
S, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 3
7: 767 (1984)). Both cell lysate and culture medium were sedimented with monoclonal antibodies specific for HA. The precipitated proteins were analyzed by SDS-PAGE.

Example 5 Cloning and Expression of Ckβ-10 Using Baculovirus Expression System DN Encoding Full-Length Ckβ-10 Protein
The A sequence, ATCC No. 75849, was amplified utilizing PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The 5 'primer has the sequence: And contains 12 nucleotides that resemble a signal that is effective for initiation of translation in eukaryotic cells (J. Mol. Biol. 198).
7, 196 , 947-950, Kozak, M.) and immediately behind are the first six nucleotides of the Ckβ-10 coding sequence (translation initiation codon “ATG” is underlined). The 3 'primer has the sequence: And contains a cleavage site for the restriction endonuclease Asp781 and 19 nucleotides complementary to the 3 'untranslated sequence of the Ckβ-10 gene. The amplified sequence was converted to a commercially available kit (“Geneclean”, BIO 101 In).
c., La Jolla, Ca.) using a 1% agarose gel. The fragment was then digested with the endonucleases BamHI and Asp781,
It was then purified on a 1% agarose gel. This fragment is named F2. Vector pRG1 (pVL941
Vector modifications (discussed below) were used for expression of the Ckβ-10 protein using the baculovirus expression system (reviewed by Summers, MD and Smi).
th, GE 1987, A manual of methods for bacul
ovirus vectors and insect cell culture procedure
s, Texas Agricultural Experimental Station Bull
letin No. 1555). The expression vector is a strong polyhedri of the Autographa California nuclear polyhedrosis virus group (AcMNPV).
n) Promoter followed by restriction endonuclease Ba
Includes recognition sites for mHI and Asp781. The simian virus (SV) 40 polyadenylation site is used for efficient polyadenylation. For easy selection of recombinant viruses, the β-galactosidase gene from E. coli is inserted in the same orientation as the polyhedron promoter followed by the polyadenylation signal of the polyhedrin gene. Viral sequences for cell-mediated homologous recombination of cotransfected wild-type viral DNA are flanked on both sides by a polyhedral sequence. Many other baculovirus vectors were constructed using pAc373, pVL
941, and could be used in place of pRG1 such as pAcIM1 (Luckow, VA;
And Summers, MD, Virology, 170: 31-3.
9). Plasmids were digested with restriction enzymes BamHI and Asp781.
And then dephosphorylated using calf intestinal phosphatase by methods known in the art. Then the DNA
Was isolated from a 1% agarose gel. This vector D
Name the NA V2. Fragment F2 and dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E. coli HB101 cells were then transformed and bacteria containing the plasmid with the Ckβ-10 gene (pBacCkβ-10) were identified using the enzymes BamHI and Asp781. The sequence of the cloned fragment was confirmed by DNA sequencing.
Using the lipofection method, plasmid pBacCK
5 μg of β-10 was added to a commercially available linearized baculovirus (“Ba
culoGold ^TM baculovirus DNA ", Pharming
en, San Diego, Calif.) with 1.0 μg (Felgner et al., Proc. Natl. Acad. Sci. U.
SA, 84: 7413-7417 (1987)). Bacu
1 μg of loGold ^™ virus DNA and plasmid
5 μg of pBacCkβ-10 was added to serum-free Grace's
Medium (Life Technologies Inc., Gaithersburg, M.M.
D) Mix in sterile wells of a microtiter plate containing 50 μl. Then, lipofectin (Lipofecti
n) 10 μl and 90 μl of Grace's medium were added, mixed and incubated at room temperature for 15 minutes. The transfection mixture is then added to 1 ml of serum-free Grace's medium,
Sf9 insect cells seeded on a 35 mm tissue culture plate (A
TCC CRL 1711). The plate was rocked back and forth to mix the newly added solution. The plate was then incubated at 27 ° C. for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was returned to the incubator and continued to be cultured at 27 ° C. for 4 days.

After 4 days, the supernatant was collected and collected with Summers and S
Plaque assays were performed as described by mith (supra). As a change, "Blue Gal" (Life Te
Agarose gel containing Chnologies Inc., Gaithersburg) was used, which allows easy isolation of blue-stained plaques. (A detailed description of the "plaque assay" is also available from the user's guide to insect cell culture, and from Life Technologies Inc.
c., Baculovirology distributed by Gaithersburg, also found on pages 9-10).

Four days after adding serial dilutions of virus to the cells, blue-stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect insect Sf9 cells seeded on 35 mm dishes. Four days later, the supernatants of these culture dishes were collected and stored at 4 ° C.

Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are transformed at a multiplicity of infection (MOI) of 2 with the recombinant baculovirus V-Ckβ
-10. Six hours later, the medium was removed and
SF900 II without methionine and cysteine
The medium was replaced with a medium (Life Technologies Inc., Gaithersburg). After 42 hours, 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S cysteine 5 μCi (Amersham) were added. After incubating the cells for an additional 16 hours, they were collected by centrifugation and the labeled proteins were visualized by SDS-PAGE and autoradiography.

Since many modifications and variations of the present invention are possible in light of the above teachings, the present invention can be practiced otherwise than as specifically described within the scope of the following claims.

Sequence Table (1) General Information: (i) Patent Applicant: Lee et al. (Ii) Title of Invention: Human Chemokine Polypeptide (iii) Number of Sequences: 4 (iv) Contact: (A) Address: Carrera, Baan, Vane, Gilfiran, Sech, Stewart and Olstein (B) Street: Becker Farm Road No. 6 (C) City: Roseland (D) State: New Jersey (E) Country: United States ( F) ZIP: 07068 (v) Computer reading / reading type: (A) Medium type: 3.5 inch diskette (B) Computer: IBM PS / 2 (C) Operating system: MS-DOS (D) Software: Word Perfect 5.1 (vi) Data of this application: (A) Application number: (B) Filing date: Same date (C) Classification: (vii) Prior art data: (A) Application number: (B) Filing date: (viii) Patent attorney / agent information: (A) Name: Ferralo, Gregory di (B) Registration number: 36,134 (C) Reference / reference number: 325800-183 (ix) Telephone contact information: (A) Telephone number: 201-994-1700 (B) Fax number: 201-994-1744 (2) Information of sequence number 1: (I) Sequence characteristics (A) Length: 291 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Type of molecule: cDNA (xi) Description of sequence: SEQ ID NO: 1 (3) Information of SEQ ID NO: 2: (i) Sequence characteristics: (A) Length: 96 amino acids (B) Type: amino acids (C) Number of chains: (D) Topology: linear (ii) Sequence Type: protein (xi) Sequence description: SEQ ID NO: 2 (4) Information of SEQ ID NO: 3: (i) Sequence characteristics (A) Length: 297 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) ) Molecular type: cDNA (xi) Description of sequence: SEQ ID NO: 3 (5) Information of SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 98 amino acids (B) Type: amino acids (C) Number of chains: (D) Topology: linear (ii) Sequence Type: Protein (xi) Sequence description: SEQ ID NO: 4

[Brief description of the drawings]

FIG. 1 shows the cDNA sequence of Ckβ-4 and the corresponding deduced amino acid sequence. The first 24 amino acids are Ckβ
Demonstrating a predicted leader sequence of -4, the predicted mature polypeptide comprises 72 amino acids. Use the standard one-letter abbreviations for amino acids.

FIG. 2 shows the cDNA sequence of Ckβ-10 and the corresponding deduced amino acid sequence. The first 23 amino acids are Ck
Demonstrating the putative leader sequence of β-10, the putative mature polypeptide comprises 75 amino acids. Use the standard one-letter abbreviations for amino acids.

FIG. 3 shows the amino acid sequence homology between Ckβ-4 and the mature peptide of eotaxin (lower).

FIG. 4: Ckβ-10 (upper) and human MCP-3 (lower)
1 shows the amino acid sequence homology between

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 1/16 48/00 11/00 A61P 1/16 11/06 11/00 17/02 11/06 17/06 17/02 19/02 17/06 31/00 19/02 35/00 31/00 35/02 35/00 37/02 35/02 37/08 37/02 43/00 105 37 / 08 C12N 15/00 ZNAA 43/00 105 A61K 37/02 (72) Inventor Haodong Lee United States 20878 Rutridge Drive, Gay Saarsburg, Maryland 11033 (72) Inventor Mark Di Adams United States 20878 Maryland No. Potomac, Dafif Drive 15205 F-term (reference) 4B024 AA01 BA21 CA04 DA02 DA05 DA11 EA02 EA03 EA04 GA11 HA01 4C084 AA02 AA13 AA17 BA01 BA08 BA20 BA21 MA01 NA14 ZA592 ZA752 ZA892 ZA962 ZB07 2 ZB112 ZB132 ZB212 ZB262 ZB272 ZB322 ZB372 4C085 AA13 AA14 CC32 EE01 GG01 4C086 AA01 AA02 EA16 MA01 MA04 NA14 ZA59 ZA75 ZA89 ZA96 ZB07 ZB11 ZB13 ZB21 ZB26 ZB27 ZB32 ZB37

Claims (1)

[Claims] 1. (a) a nucleic acid encoding a polypeptide comprising the entire amino acid sequence shown in FIG. 1 or FIG. 2; (b)) at least the mature form of the polypeptide having the deduced amino acid sequence shown in FIG. 1 or FIG. (C) a nucleic acid having the coding sequence shown in FIG. 1 or 2 that encodes at least the mature form of the polypeptide; (d) a nucleic acid according to any of (a) to (c) A nucleic acid encoding a fragment, analog, or derivative of the encoded polypeptide; (e) its complementary strand hybridizes under stringent conditions to any of the nucleic acids (a) to (d), and Ckβ-
(F) a nucleic acid encoding a polypeptide having 4 activities; (f) its complementary strand hybridizes under stringent conditions to any of the nucleic acids (a) to (d), and
(G) a nucleic acid that encodes a polypeptide having 10 activities; (g) the nucleic acid does not encode an N-terminal methionine; any nucleic acid of (a) to (f); (h) the nucleic acid encodes an N-terminal methionine (A)
(I) a polynucleotide complementary to the nucleic acid of any of (a) to (h); (j) ATCC Deposit No. 75848 or 7584
No. 9 nucleic acid encoding a polypeptide comprising the entire amino acid sequence encoded by the cDNA; (k) ATCC Deposit No. 75848 or 7584
No. 9 nucleic acid encoding a polypeptide comprising at least the mature form of the polypeptide encoded by the cDNA; (1) A encoding at least the mature form of the polypeptide
A nucleic acid having a cDNA coding sequence contained in TCC Deposit No. 75848 or 75849; (m) a fragment, analog or derivative of a polypeptide encoded by any of the nucleic acids (j) to (l); An encoding nucleic acid; (n) its complementary strand hybridizes under stringent conditions to any of the nucleic acids (j) to (l), and
(O) a nucleic acid encoding a polypeptide having four activities; (o) its complementary strand hybridizes under stringent conditions to any of the nucleic acids (j) to (m), and
(P) the nucleic acid does not encode an N-terminal methionine; any nucleic acid of (j) to (o); (q) the nucleic acid encodes an N-terminal methionine (J)
(R) a polynucleotide complementary to the nucleic acid of any of (j) to (q); a polynucleotide comprising a nucleic acid selected from the group consisting of: For protection treatment during cancer chemotherapy of a polypeptide comprising an amino acid sequence encoded by leukemia, solid tumor, chronic infection, autoimmune disease, psoriasis, asthma, allergy, or wound healing Use to promote.